Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on β2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.


Supplementary Notes 1-11
Supplementary Figures 1-14 Supplementary Tables 1-5 Supplementary References positive aggregates formed by ultrasonication showed a weak-contrast fibrillar morphology ( Supplementary Fig. 4a), and the aggregates seeded and elongated from the amyloid fibrils formed by ultrasonication showed a clear-contrast fibrous morphology ( Supplementary Fig. 4b). The ThT-negative aggregates formed in the presence of 15% (v/v) serum were amorphous ( Supplementary Fig. 4c). The serum concentrations of 2m monomer were clearly higher in the serum of dialysis patients than those of non-dialysis controls ( Supplementary Fig. 5a). The concentrations in dialysis patients and non-dialysis controls were 23.9 ± 5.2 (N = 30) and
Meanwhile, the serum concentrations of 2m monomer markedly decreased after maintenance dialysis treatments ( Supplementary Fig. 5b). The pre-and post-dialysis 2m concentrations were 24.9 ± 5.3 (N = 28) and 7.7 ± 4.4 g/mL (N = 28), respectively, which were also consistent with the literature 1,2 . When serum was added to the recombinant 2m solution at a concentration of 5% (v/v), the concentration of 2m monomer from sera was at most 2 g/mL, being much less than in standard solutions (1.0 mg/mL) for the HANABI assay, and its effects on amyloid fibril formation would be negligible.

Supplementary Note 4: Relationship between ThT fluorescence intensity and the amount of amyloid fibrils in the presence of sera
In amyloid fibril formation, the addition of preformed fibrils bypasses the primary nucleation and accelerates amyloid formation, the so-called seeding reaction 3 . In general, the reaction time for amyloid formation is shortened in a seed-concentrationdependent manner, i.e., the higher seed concentrations induce more rapid amyloid fibril formation 4 . Based on the concentration-dependent manner of the seeding reaction, we qualitatively evaluated the relationship between the ThT fluorescence intensity of amyloid fibrils formed at various serum concentrations and the net amount of amyloid fibrils.
As shown in Supplementary Fig. 6a, b, the lag time of amyloid formation depended on the seed concentration, indicating that the shorter the lag time, the greater the amount of preformed amyloid fibrils in the seed solution. Next, the seeds formed at serum concentrations of 0-10% (v/v) were added to the monomer solution, and the concentration of amyloid fibrils was evaluated by the seeding reaction, as shown in Supplementary Fig. 6c. As summarized in Supplementary Fig. 6d, the higher serum concentration led to a longer lag time in the seeding reaction. Supplementary Fig. 6e shows the relationship between ThT fluorescence intensity and the lag time in the seeding reaction. A shorter lag time indicates a higher amyloid fibril concentration in the sample solution, which means that the fibril concentration in the sample solution positively correlates with ThT fluorescence intensity of the sample solution.
Supplementary Note 5: Examination of significance of change in the effects of serum on amyloid formation before and after maintenance dialysis treatments To examine the change in the effects of serum on amyloid fibril formation before and after maintenance dialysis treatment, the ThT fluorescence assay with ultrasonication was performed using sera collected from 28 patients immediately before and after maintenance dialysis treatments. ThT kinetics are shown in Supplementary Fig.   8a, where red and blue lines denote the results with sera collected before and after the treatment, respectively. For each sample, ThT kinetics were measured using multiple independent solutions (n ≥ 4). Using the data, the significance of any change in the lag time and ThT fluorescence intensity between sera collected before and after maintenance dialysis treatments was investigated by the unpaired one-sided t-test, as shown in Supplementary Fig. 8b, c, respectively. In 28 patients examined, 16 and 23 patients showed significantly slower kinetics (lag time) and less resultant amyloid fibrils (ThT fluorescence intensity) after the maintenance dialysis treatments, indicating that the inhibitory effects of serum on fibril formation improved after a single dialysis treatment in more than half of the dialysis patients (Supplementary Table 1 Tables 3 and 4. Among them, we focused on five serum components that showed correlation coefficients of 0.5 or higher for both the lag time and ThT fluorescence intensity: 2m monomer, blood urea nitrogen, creatinine, ureic acid, and serum albumin. Here, it should be noted that the final concentration of 2m monomers was negligible because it was less than 1% of the concentration of recombinant 2m monomers included in the standard solution used in the HANABI assays. On the other hand, blood urea nitrogen is an indicator of the blood concentration of urea. Urea is a classical denaturant of native proteins 5 , shifting the equilibrium from native folded monomers to denatured unfolded monomers in a concentration-dependent manner. We previously studied the urea-induced unfolding of 2m monomers under a neutral condition 6 . The results showed that the urea concentration at the midpoint of denaturation was ~5 M. The serum concentration of blood urea nitrogen was less than 100 mg/dL (Supplementary Fig. 9, panel 25), which corresponds to a urea concentration of less than 36 mM. Such a low-concentration urea fails to affect the 2m monomer stability and its amyloid fibril formation. Therefore, since the high correlation between these two serum components and amyloidogenecity can be attributed to a spurious correlation, these two serum components were excluded as candidates for factors affecting amyloid formation.
Effects of three other serum components: serum albumin, ureic acid, and creatinine, on 2m amyloid fibril formation were investigated, as shown in Supplementary   (1) and (2)  (iii) Temperature dependence: To include the temperature effects into the unified model, we considered the temperature dependences of three equilibrium constants, KD C , KF, and KP. Regarding KD C , the QCM results (Fig. 5d, e, and Supplementary Fig.   11) showed that KD C remained on the same order of magnitude regardless of the reaction temperature. Then, KD C was assumed to be independent of temperature and was used the value of KD C = 500 M.

Supplementary Note 11. Estimation of the onset risk of DRA
According to the classical nucleation theory 11 , the nucleation rate of a solute in a supersaturated solution, J, is denoted as: where ‡ , R, and T denote the activation free energy, gas constant, and absolute temperature, respectively. It is known that the ‡ value depends on the supersaturation ratio, , written as 11 ‡ ∝ ln −2 ( ). (S16) In the amyloid fibril formation, the fibrils are formed by aggregation of the supersaturated denatured monomers. Thus, in this study, the  value is denoted as: Given that the time for nucleation, nuc. , is inversely proportional to the nucleation rate 12 , the following equation is derived: ln( nuc. ) ∝ ln( −1 ) ∝ ln −2 ( ).
( > 1) (S18) It should be noted that the nucleation reaction never occurs when ≤ 1. Here, we replaced nuc. as a lag time for amyloid fibril formation because amyloid fibrils rapidly grow once nuclei form. These relations were used to estimate the onset risk of DRA.
The fluctuation of the temporary risk of amyloid fibril formation with time was calculated based on the change in the total 2m and albumin concentrations with time, as: